Nucleic acid molecules encoding enzymes having fructosyltransferase activity, and their use

ABSTRACT

Nucleic acid molecules are described which encode polypeptides having the enzymatic activity of a fructosyltransferase. Also, vectors, host cells and transgenic plants are described which contain such nucleic acid molecules. Furthermore, processes for producing polyfructose, particularly that of the inulin type, using the hosts described and/or the fructosyltransferase produced by them are described.

The present invention relates to nucleic acid molecules encodingpolypeptides having the enzymatic activity of a fructosyltransferase.The invention furthermore relates to vectors and hosts containing suchnucleic acid molecules. The present invention also relates to processesfor producing fructosyltransferase and the production of polyfructosesof the inulin type in various host organisms or in vitro, as well as thefructosyltransferase encoded by the nucleic acid molecules describedwhich may be used to produce polyfructoses of the inulin type.

Water-soluble, linear polymers can be put to various uses, for examplesfor increasing the viscosity of aqueous systems, as detergents, assuspension agents or for sedimentation acceleration and complexing, butalso to bind water. Saccharide-based polymers, for example fructosylpolysaccharides, are particularly interesting starting materials as theyare biologically degradable.

In addition to their use as renewable raw materials in industrialproduction and manufacture, fructosyl polymers are attractive as foodadditives, for example as sweeteners. Polymers of various chain lengthsare required for the various applications. While for the food sectorpolymers of short or medium chain length are preferred, technicalapplications, for example the production of surfactants, requirepolymers with a high degree of polymerization (DP).

Various processes have been described for producing fructanpolysaccharides in plants by expressing fructosyltransferases ofbacterial origin or for producing polyfructoses of medium chain lengthby expressing fructosyltransferases of plant origin. PCT/US89102729,e.g., describes the possibility of generating carbohydrate polymers,particularly dextrane or polyfructose, in transgenic plants, namelyspecifically in the fruit of transgenic plants. In order to generateplants that are modified in that way it is proposed to use levan sucrosefrom microorganisms, particularly from Acrobacter levanicum,Streptococcus salivarus and Bacillus subtilis, or of dextrane sucrasesfrom Leuconostoc mesenteroides. Neither the generation of the activeenzyme, nor that of levan or dextrane nor the production of transgenicplants is described.

PCT/EP93/02110 discloses a method for producing transgenic plants whichexpress the Isc gene of the levan sucrase from the gram-negativebacterium Erwinia amylovora. The plants produce a high-molecular, highlybranched levan. PCT/NL93/00279 describes the transformation of plantswith chimeric genes that contain the sacB gene from Bacillus subtilis.Such plants produce a branched levan. The bacterialfructosyltransferases used in PCT/US89/02729, PCT/EP93/02110 andPCT/NL93/00279 synthesize levan, a β-2,6 linked fructosyl polymer whichhas numerous β-2,1 branchings. Due to the numerous branchings, however,levan involves decisive disadvantages for the technical processing andis therefore much less in demand as technical starting material thaninulin which displays β-2,1 linkings. Presently, only one bacterial geneis known the gene product of which is involved in the synthesis ofinulin, said gene being the ftf gene from Streptococcus mutans.PCT/NL93/00279 describes the transformation of plants with said genewhich synthesize high-molecular inulin but in such small amounts that itcannot be economically utilized. PCT/EP97/02195, too, describes aprocess for producing transgenic, inulin-producing plants with the ftfgene from Streptococcus mutans. Like with the plants described inPCT/NL93/00279 the yield of high-molecular inulin is low. While it ispossible to express the gene in plants if the gene was geneticallyengineered beforehand, the yield in inulin that can be obtained fromtransgenic plants is so low that the transgenic plants cannot beeconomically utilized. Furthermore, PCT/NL96/00012 discloses DNAsequences which encode carbohydrate polymer-synthesizing enzymes as wellas the production of transgenic plants using said DNA sequences. Thesequences disclosed originate from Helianthus tuberosus. In accordancewith PCT/NL96/00012 the sequences disclosed can be used to modify thefructan profile of petunia and potato but also of Helianthus tuberosusitself. Expression of the sequences disclosed which encode asucrose-dependent sucrose fructosyltransferase (SST) or a fructanfructosyl transferase in transgenic plants allows the production ofinulin. The average polymerization degree of the inulin is, however,DP=6 to DP=10. With such a polymerization degree the inulin cannot beconsidered long-chain. The process described in PCT/NL96/00012 does notallow to produce high-molecular inulin. Recently, Rehm et al. (J.Bacteriology 180 (1998), 1305-1310) reported the generation ofoligosaccharides in yeast by introducing an SST from Aspergillusfoetidus. However, the polymerization degree of the product obtained wasonly DP=3.

DE 197 08 774.4 relates to the production of 1-kestose and nystose usingenzymes having fructosyl polymerase activity. The tri- andtetrasaccharide may be produced in transgenic plants. The yield is highand in potato corresponds to the cellular content of sucrose. However,the production of longer-chain inulin is not described. The synthesis ofpolyfructoses by fungi is also discussed in many publications.Barthomeuf and Pourrat (Biotechnology Letters 17 (1995), 911-916),describe, e.g., an enzyme preparation of Penicillium rugulosum which hasfructosyltransferase activity. The preparation exhibits variousenzymatic properties and therefore does not represent a purefructosyltransferase. DNA sequences of the fructosyltransferase gene arenot known. Cairns et al. (New Phytologist 129 (1995), 299-308) describea transient synthesis of tri-, tetra- and pentasaccharides from sucrosein the culture medium of Monographella nivalis. The underlying enzymaticactivity appears to be of mainly hydrolytic nature since thepolyfructoses are degraded again by the enzyme with increasing substrateexhaustion. Since no DNA sequence is known it is not possible toassess—relying on the homology with fructofuranosidases (invertases) asreference—whether a fructosyltransferase in the proper sense or aninvertase is present.

It was shown for the fungus Aspergillus sydowi IAM 2544 that it iscapable of generating polyfructoses of the inulin type. Harada et al.(in: Nishinari and Doi (Eds.), Food Hydrocolloids: Structures,Properties and Functions, Plenum, N.Y. (1994), 77-82) describe, forexample, the synthesis of inulin with conidia of Aspergillus sydowi. 125g conidia were incubated in 25 l 20% sucrose solution. The productgenerated was purified by HPLC. However, such a process does not lenditself for a large-scale production of inulin. Maramatsu et al. (Agric.Biol. Chem. 52 (1988), 1303-1304) describe the production offructooligosaccharides with mycelium of the same fungal strain (A.sydowi IAM 2544). The polymerization degree is reported to be 3 to 13.The enzymes involved in this process were not or only partiallypurified. Amino acid sequences or DNA sequences of the correspondinggenes are not known. Instructions for the purification of the proteinsare not or only incompletely given.

The problem underlying the present invention is therefore to providenucleic acid molecules and processes allowing to produce geneticallyengineered organisms that are capable of generating polyfructoses of theinulin type.

This problem is solved by providing the embodiments characterized in theclaims.

The invention thus relates to nucleic acid molecules encoding afructosyltransferase, selected from the group consisting of

-   (a) nucleic acid molecules encoding a protein comprising the amino    acid sequence indicated in SEQ ID No. 2;-   (b) nucleic acid molecules comprising the nucleotide sequence of the    coding region indicated in, SEQ ID No. 1 or a corresponding    ribonucleotide sequence;-   (c) nucleic acid molecules hybridizing to a strand complementary to    the nucleic acid molecules indicated in (a) or (b); and-   (d) nucleic acid molecules the nucleotide sequence of which deviates    from the sequence of the nucleic acid molecules mentioned in (c) due    to the degeneracy of the genetic code;    as well as nucleic acid molecules which are complementary thereto.

The sequence depicted in Seq ID No. 1 encodes a sucrose-dependentfructan fructosyltransferase from Aspergillus sydowi which leads inplant cells to the synthesis of a long-chain polyfructan of the inulintype. It was surprisingly found that it is possible to producelong-chain polyfructans of the inulin type in high yields in hostorganisms, specifically in transgenic plants, bacteria or fungi whenusing said sequences.

Within the scope of the present invention instructions for thepurification of the enzyme from Aspergillus sydowi were elaborated. Theenzyme was purified up to homogeneity so as to be able to detect aminoacid sequences. On the basis of the sequence information obtainedprimers for a polymerase chain reaction were detected. Gene fragmentswere amplified with the help of these primers which were used forscreening cDNA libraries. Several cDNA molecules with sequence homologyto the PCR products were prepared and compared. Most of the cDNAmolecules obtained had inserts of the same size. Completeness of thecDNA molecules was confirmed upon functional expression of the DNAsequences in Saccharomyces or in potato.

The purification of the enzymes, the design of primers for PCR, theidentification of cDNA molecules and the heterologous expression aredescribed in the examples. The isolated DNA sequence and its derivableprotein sequence are depicted in Seq. ID No 1 and 2, respectively. TheDNA sequence according to the invention is the first to encode asucrose-dependent fructan fructosyltransferase from fungi. The DNAsequence and the protein sequence encoded by it differ widely from DNAsequences encoding already known fructosyltransferases. For example,there is only a 22.6% and 39% identity with the fructosyltransferasefrom Aspergillus naeslundii lev j on the protein and DNA level,respectively. While there is a 64 and 60.6 identity on the protein andDNA level, respectively, with an invertase gene from Aspergillus niger,the protein encoded by said gene has a completely different enzymeactivity. This goes to show that the nucleic acid molecules and thefructosyltransferases encoded by them are molecules that have not beendescribed so far.

In the context of the present invention a fructosyltransferase isunderstood to be a protein which is capable of catalyzing the linkage ofβ-2,1-glycosidic and/or β-2,6-glycosidic bonds between fructose units.The fructosyl residue to be transferred originates from sucrose.

The reaction catalyzed by a sucrose-dependent fructanfructosyltransferase according to the invention can be depicted asfollows:n(G−F)→G−F_(n)+(n−1)G

In this scheme G=glucose, F=fructose and G−F=sucrose. I.e., the enzymetransfers fructose residues from sucrose to a polyfructan which isformed starting from a sucrose molecule by β-2,1-glycosidic links,wherein β-2,6 glycosidic links may also occur.

A polypeptide encoded by a nucleic acid molecule according to theinvention with the activity of a fructosyltransferase leads to thesynthesis of polyfructose and particularly in plant cells to thesynthesis of polyfructose of the inulin type (hereinafter also referredto as inulin).

In the context of the present invention, polyfructose is understood tobe a polyfructan with a polymerization degree of DP≧4, preferably ofDP≧6, and specifically of DP≧8. “Polyfructose of the inulin type” or“inulin” is intended to refer to a long-chain fructan polymer, themolecules of which are mainly β-2,1-glycosidically linked and optionallyalso have β-2,6 branches. The term “long-chain” means that thepolymerization degree (DP) is more than 20, preferably more than 50,more preferably more than 100 and most preferred more than 200. Thefructosyl polymer may bear a terminal glucose residue which is linkedvia C-1 OH group of the glucose and the C-2 OH group of a fructosylresidue. In this case, one molecule sucrose is contained in thefructosyl polymer.

When the enzyme is used in vitro for the synthesis of polyfructan, anoligomer product is obtained (DP=4 to 10).

The enzyme encoded by the nucleic acid molecules according to theinvention may be particularly distinguished from knownfructosyltransferases due to the catalyzed reaction. For example, theknown plant SSTs catalyze the reaction:G−F+G−F→G−F−F+G.

The fructan-dependent fructan-fructosyltransferases from plants,however, catalyze the reaction:G−F_(n)+G−F_(m)→G−F_(n+1)+G−F_(m−1),this reaction being completely reversible.

Bacterial fructosyltransferases, e.g., that encoded by the sacB genefrom Bacillus subtilis, also catalyze a reaction of the typenG−F→G−F_(n)+(n−1)G.

However, these enzymes produce levan, i.e., a β-2,6-glycosidicallylinked polyfructan with β-2,1-branching.

While the protein (an FTF) encoded by the ftf gene from Streptococcusmutans induces the synthesis of inulin with a molecular weight ofseveral million Daltons. However, the ftf gene or the encoded proteindoes not exhibit an appreciable homology to the nucleic acid sequencedepicted in SEQ ID No. 1 (only 37.3%) or the amino acid sequencedepicted in SEQ ID No. 2 (only 22.6%).

In the context of the present invention, the term “hybridization” meanshybridization under conventional hybridization conditions, preferablyunder stringent conditions, such as described in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. An example ofstringent hybridization conditions is the hybridization in 50%formamide, 5×SSC, 5× Denhardt's solution, 40 mM sodium phosphate pH 6.8;0.5% (wt./vol.) BSA, 1% (wt./vol.) SDS, 0.1 mg/ml herring sperm DNA at ahybridization temperature of 42° C. and subsequently washing the filtersin 0.5×SSC/0.5% SDS at 60° C.

An example of conventional non-stringent hybridization conditions is ahybridization under the conditions mentioned above with the exceptionthat 30% formamide are used instead of 50% and the filters aresubsequently washed in 2×SSC/0.5% SDS at 56° C. Nucleic acid moleculeshybridizing to the molecules according to the invention can be isolatedfrom, e.g., genomic or cDNA libraries which are preferably prepared fromfungi. Such nucleic acid molecules can be identified and isolated usingthe molecules according to the invention or fragments of these moleculesor the reverse complements of these molecules, e.g., by hybridizationaccording to standard methods (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Nucleic acid molecules maybe used as hybridization probe that exhibit exactly or substantially thesame nucleotide sequence depicted in SEQ ID No. 1 or fragments of thissequence. The fragments used as hybridization probe may also besynthetic fragments that were produced using conventional techniques ofsynthesis and the sequence of which is substantially identical to thesequence of a nucleic acid molecule according to the invention.

The molecules hybridizing to the nucleic acid molecules according to theinvention also comprise fragments, derivatives and allelic variants ofthe nucleic acid molecules described above that encodes a proteinaccording to the invention. “Fragments” are understood to be parts ofthe nucleic acid molecules that are long enough to encode a protein withfructosyltransferase activity. The term “derivative” as used in thepresent invention means that the sequences of these molecules differfrom the sequences of the nucleic acid molecules described above in oneor several positions but exhibit a high degree of homology to thesesequences. Homology means a sequence identity of at least 40%,particularly an identity of at least 60%, preferably of more than 80%,and even more preferred of more than 90%. The proteins encoded by thesenucleic acid molecules preferably have a sequence identity to the aminoacid sequence indicated in SEQ ID No. 2 of at least 60%, preferably ofat least 70%, particularly of at least 80%, particularly preferred of atleast 90%, and most preferred of at least 95%. The deviations from thenucleic acid molecules described above may be brought about by, e.g.,deletion, substitution, insertion and/or recombination. Nucleic acidmolecules according to the invention may be other derivatives of thesequences of fungal origin. A derivatization of the sequences may berequired so as to facilitate expression in certain host cells.

The nucleic acid molecules that are homologous to the moleculesdescribed above and represent derivatives of said molecules areregularly variations of these molecules that represent modificationsexerting the same biological function. These variations may be eithernaturally occurring, for example sequences from other strains ororganisms, or mutations. These mutations may have occurred naturally ormay have been introduced by specific mutagenesis. Also, the variationsmay be synthetically produced sequences. The allelic variants may beeither naturally occurring variants or synthetically produced orgenerated by recombinant DNA techniques.

The proteins encoded by the various variants of the nucleic acidmolecules according to the invention preferably have certaincharacteristics in common such as enzymatic activity, molecular weight,immunological reactivity or conformation or physical properties as theelectrophoretic mobility in gel electrophoreses, chromatographicbehavior, sedimentation coefficient, solubility, spectroscopicproperties, stability, pH optimum or temperature optimum.

In a preferred embodiment, the nucleic acid molecules according to theinvention encode a polypeptide having, the properties of a fungalfructosyltransferase, particularly preferred from Aspergillus and mostpreferred of a fructosyltransferase from Aspergillus sydowi.

The nucleic acid molecules according to the invention may be either DNAor RNA molecules. Examples of corresponding DNA molecules are genomicDNA or cDNA molecules. The nucleic acid molecules according to theinvention can be isolated from natural sources, for example from fungi,particularly Aspergillus and preferably from Aspergillus sydowi, or theymay be synthesized according to known methods. It is also possible tointroduce various mutations into the nucleic acid molecules according tothe invention by way of conventional techniques of molecular biology(see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual,2^(nd) edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Such an introduction induces the synthesis of proteinswith potentially modified biological properties. One approach is togenerate deletion mutants in which nucleic acid molecules are generatedby progressive deletions at the 5′ or at the 3′ end of the coding DNAsequence that lead to the synthesis of correspondingly truncatedproteins. Another approach is to specifically produce enzymes that arelocalized in various compartments due to the addition of signalsequences. In order to achieve a localization of the proteins accordingto the invention in the cytosol, no signal sequences have to be added tothe sequence indicated in SEQ ID No. 2.

On the other hand, point mutations might also be introduced at positionswhere a modification of the amino acid sequence influences, for example,the enzyme activity or the regulation of the enzyme. In this way, e.g.,mutants with a modified K_(m)-value may be produced, or mutants whichare no longer subject to the regulation mechanisms by allostericregulation or covalent modification usually occurring in cells.

Furthermore, mutants with a modified substrate or product specificitymay be produced. Moreover, mutants with a modifiedactivity-temperature-profile may be produced.

For the genetic manipulation in prokaryotic cells the nucleic acidmolecules of the invention or fragments of these molecules may beintegrated into plasmids which allow for a mutagenesis or a sequencemodification by recombination of DNA sequences. By means of standardmethods (cf. Sambrook et al., Molecular Cloning, A Laboratory Manual,2^(nd) edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) base exchanges may be carried out or natural or syntheticsequences may be added. In order to link the DNA fragments, adapters orlinkers may be attached to the fragments. Moreover, use can be made ofmanipulations which offer suitable restriction sites or which removesuperfluous DNA or restriction sites. Wherever inserts, deletions orsubstitutions may be useful, in vitro mutagenesis, “primer repair”,restriction or ligation may be used. For analysis, use is generally madeof a sequence analysis, a restriction analysis or furtherbiochemico-molecularbiological methods.

Furthermore, the invention relates to vectors which contain the nucleicacid molecules according to the invention. Preferably, these vectors areplasmids, cosmids, viruses, bacteriophages and other vectors common ingenetic engineering.

Preferably, the nucleic acid molecule according to the invention isoperably linked in the vector according to the invention with regulatoryelements which allow transcription and synthesis of a translatable RNAin pro- and/or eukaryotic cells. For example a vector according to theinvention contains the following elements:

-   1. A promoter guaranteeing the transcription of downstream coding    regions in cells of the host organism, and optionally enhancer    elements.-   2. A coding region fused to the promoter which contains at least one    open reading frame for the translation of a polypeptide. In the    present invention, the coding region is a nucleic acid molecule    according to the invention.-   3. Optionally additional sequences fused to the coding region, for    example transcription termination signals, if these are required for    a successful gene expression in a certain host organism, or signal    sequences influencing the subcellular localization of the gene    product or inducing secretion of the protein.

Such a vector may contain additional genes such as marker genes allowingselection of the vector in a suitable host cell and under appropriateconditions. Expression of the nucleic acid molecule according to theinvention includes the transcription of the nucleic acid molecule into atranslatable mRNA. Regulatory elements allowing the expression of thenucleic acid molecule in prokaryotic and eukaryotic cells are known tothe person skilled in the art. Possible regulatory elements which aresuitable for the expression of the nucleic acid molecule according tothe invention in prokaryotic host cells include, for example, the P_(L),lac, trp or tac promoter in E. coli. It is particularly preferred to usethe lacZ promoter which is inducible in E. coil IPTG. Examples ofregulatory elements allowing the expression in eukaryotic host cells arethe AOX1 and the GAL1 promoter in yeast or the CMV SV40, RSV promoter,CMV enhancer, SV40 enhancer or a globin intron in mammalian cells orother animal cells. For the expression in yeast, the promoter of thealcohol dehydrogenase gene from Saccharomyces cerevisiae is preferablyused which is highly active in yeast. Further suitable vector systemshave been described in the prior art, for example in Sambrook, MolecularCloning, A Laboratory Manual (1989), Cold Spring Harbor LaboratoryPress, N.Y., and in Ausubel, Current Protocols in Molecular Biology(1989), Green Publishing Associates and Wiley Interscience, N.Y.

Regulatory elements for the expression of the nucleic acid moleculeaccording to the invention in plant cells is in principle any promoter,enhancer, terminator, etc., which is active in plant cells. The promotermay be selected in such a way that the expression takes placeconstitutively or in a certain tissue, at a certain point of time of theplant development or at a point of time determined by externalcircumstances. With respect to the plant the promoter may be, homologousor heterologous.

Suitable promoters for a constitutive expression are, e.g., the 35S RNApromoter of the Cauliflower Mosaic Virus (see, e.g., U.S. Pat. No.5,352,605) and the ubiquitin promoter (see, e.g., U.S. Pat. No.5,614,399) for a constitutive expression, the patatin gene promoter B33(Rocha-Sosa, EMBO J. 8 (1989), 23-29) for a tuber-specific expression inpotato, or a promoter which ensures expression only inphotosynthetically active tissues, e.g., the ST-LS1 promoter.(Stockhaus, Proc. Natl. Acad. Sci. USA-84 (1.987), 7943-7947; Stockhaus,EMBO J. 8 (1989), 2445-2451), the Ca/b promoter (see, e.g., U.S. Pat.No. 5,656,496, U.S. Pat. No. 5,639,952, Bansal, Proc. Natl. Acad. Sci.USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (see, e.g., U.S.Pat. No. 5,034,322, U.S. Pat. No. 4,962,028). However, promoters canalso be used that are activated only at a certain point of timedetermined by external circumstances (see, e.g., WO 93/07279). Promotersof heat-shock proteins allowing a simple induction can be of specificinterest. Also, seed-specific promoters, such as the USP promoter ofVicia faba, can be used which allows a seed-specific expression in Viciafaba and other plants (Fiedler, Plant Mol. Biol. 22 (1993), 669479;Bäumlein, Mol. Gen. Genet. 225 (1991), 459-467). Furthermore,fruit-specific promoters can be used such as described in WO 91/01373.For an expression in ripening tomato fruit, e.g., cis-regulatoryelements of a polygalacturonase promoter from tomato are suitable whichare active in the outer or inner pericarp (Nicholass et al., Plant Mol.Biol. 28 (1995), 423-435; Montgomery et al., Plant Cell 5 (1993),1049-1062). Another fruit-specific promoter for tomato is described byVan Haaren et al. (Plant Mol. Biol. 21 (1993), 625-640).

Furthermore, promoters for an endosperm-specific expression may be used,such as the glutelin promoter (Leisy, Plant Mol. Biol. 14 (1990), 41-50;Zheng, Plant J. 4 (1993), 357-366), the HMG promoter of wheat, the USPpromoter, the phaseolin promoter or promoters of zein genes of maize(Pedersen, Cell 29 (1982), 1015-1026; Quattrocchio, Plant Mol. Biol. 15(1990), 81-93) or the shrunken-1-promoter (sh-1) of maize (Werr, EMBO J.4 (1985), 1373-1380).

The expression of the nucleic acid molecules according to the inventionis particularly advantageous in those parts of the plant which have anincreased sucrose content or which store sucrose. Such organs are, e.g.,the root of the sugar beet or the stem of sugar cane and sweet sorghum.Particularly preferred are therefore promoters mediating expression inthese organs, such as the patatin promoter B33 of Solanum tuberosum. Forthe specific expression in the stem of sugar cane, the ubiquitinpromoter in combination with the first intron may be used. The vectorsaccording to the invention may also possess additional functional unitswhich effect a stabilization of the vector in a host organisms, such asa bacterial replication origin or the 2-micron-DNA for the stabilizationand autonomous replication in yeast. Also, “left border” and “rightborder” sequences of agrobacterial T-DNA may be contained which allows astable integration of the genome of plants. Also, a termination sequencemay be present which serves to correctly terminate the transcription oradd a poly-A tail to the transcript which is said to have a function inthe stabilization of the transcripts. Such elements are described in theliterature (cf., e.g., Gielen, EMBO J. 8 (1989), 23-29) and are freelyexchangeable. In a preferred embodiment, the nucleic acid moleculecontained in the vector according to the invention comprises a regionwhich contains a functional signal sequence for the secretion of theencoded enzyme. Such sequences are known. An example of a signal peptideallowing localization of the protein in the vacuole is the signalpeptide of the carboxypeptidase Y from yeast (CPY). The correspondinggene has been described in, e.g., Valls et al. (Cell 48, 887-899). Plantsignal sequences are, e.g., those of the lectin genes from barley(Raikhel and Lerner, Dev. Genet. 12 (1991), 255-269) or the 43 aminoacids from the N-terminal region of the mature phytohemagglutinin ofbeans (Tague et al., Plant Cell 2 (1990), 533-546). An example of aC-terminal signal peptide is that of chitinase. (Neuhaus et al., PlantJ. 5 (1994), 45-54).

A preferred signal sequence is, e.g. the signal sequence of theproteinase inhibitor II gene from potato. However, any other signalsequence leading to secretion of a polypeptide in the chosen host may beused. The secreted fructosyltransferase may be obtained from the culturemedium and used for in vitro syntheses.

In a particularly preferred embodiment, the nucleic acid moleculecontained in the vector contains a region which encodes a signalsequence for the localization in the vacuole of plant cells, preferablythat of the patatin gene from potato (Sonnewald, Plant. J. 1 (1998),95-106). This allows the subcellular localization of thefructosyltransferase in the vacuoles of genetically engineered plantcells and plants, for example sugar beet or potato, and the accumulationof high molecular polyfructoses of the inulin type in the vacuoles.Further vacuole-located signal sequences have been described, e.g., byMatusoaka (Journal of Experimental Botany 50 (1999), 165-174),Chrispeels (Cell 68 (1992), 613-616), Matsuoka (Proc. Natl. Acad. Sci.USA 88 (1991), 834-838), Bednarek (Plant Cell 3 (1991), 1195-1206),Nakamura (Plant Phys. 101 (1993), 1-5).

In another embodiment of the invention, the nucleic acid moleculecontained in the vector comprises a region which encodes a signalsequence for the plastidic localization in plant cells.

As signal sequence, for example, the signal sequence of theferrodoxin:NADP(+)-oxidoreductase (FNR) of spinach may be used. Thesequence contains the 5′ non-translated regions as well as the flankingtransit peptide sequence of the cDNA of the plastidic proteinferrodoxin:NADP(+)-oxidoreductase (FNR) of spinach (nucleotide −171 to+165; Jansen, Current Genetics 13 (1988), 517-522).

Also, the transit peptide of the waxy protein of maize plus the first 34amino acids of the mature waxy protein (Klösgen, Mol. Gen. Genet. 217(1989), 155-161) may be used as signal sequence. In a preferredembodiment of the invention, the transit peptide of the waxy protein ofmaize is used without the first 34 amino acids of the mature waxyprotein.

In a particularly preferred embodiment, the invention relates toplasmids pSK-as1, p112-as1, pA7-as1, p35-as1, p35-s3-as1, theconstruction of which is described in the Examples (FIG. 1 to 5).

The nucleic acid molecules and expression vectors according to theinvention allow the production of polyfructoses of the inulin type invarious host organisms, particularly in plants, fungi and bacteria. Theencoded enzymes may be also used outside the host organisms for theproduction of polyfructoses of the inulin type. I.e., it is particularlypossible to use the nucleic acid molecules according to the inventionfor the production of the proteins encoded by them in any host cells, toobtain the protein from the host cells and/or the culture medium and touse it for the in vitro synthesis of inulin.

For example, a construct which contains the alcohol dehydrogenasepromoter and a nucleic acid molecule according to the invention may beused for the transformation of Saccharomyces cerevisiae. Since yeastsare not capable of taking up sucrose, cells should be used that expressa sucrose transporter due to the introduction of a heterologous DNAsequence. The production of such cells has been described, e.g., inRiesmeier et al. (EMBO J. 11 (1992), 4705-4713). Yeasts transformed withthe above-described construct form long-chain polyfructoses of theinulin type. Since the fructosyltransferase of Aspergillus sydowi doesnot possess a signal peptide, it is not secreted. The long-chainpolyfructoses are therefore generated in the yeast cells. Yeast cellscontaining these polyfructoses may be directly used as food additives.If the polyfructoses are to be obtained fermentatively in the culturemedium, a signal sequence may be fused to a nucleic acid moleculeaccording to the invention for secretion.

In another embodiment, the invention relates to host cells thattransiently or stably contain the nucleic acid molecules or vectorsaccording to the invention or, that are derived from such a cell. Theterm “host cell” relates to an organism that is capable of taking up invitro recombined DNA and optionally synthesizing the proteins encoded bythe nucleic acid molecules according to the invention. The host cellsmay be either of prokaryotic or eukaryotic origin. The term“prokaryotic” includes all bacteria that can be transformed ortransfected with a nucleic acid molecule according to the invention andthat advantageously allow the expression of a protein havingfructosyltransferase activity. Prokaryotic host cells include, e.g.,both gram-negative and gram-positive bacteria, such as E. coli, S.typhimurium, Serratia marcescens and Bacillus subtilis. The term“eukaryotic” includes insect cells, fungal cells, plant cells, animaland human cells. Preferred fungal cells are, e.g., those that are or maybe used for fermentation, particularly Saccharomyces, particularlypreferred S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia etc.Preferably, such a fungal cell is a cell from the genus Aspergillus andparticularly preferred from the species Aspergillus niger. Of particularinterest is the expression of the fructosyltransferase according to theinvention in these cells in combination with a secretory signalsequence, e.g., that of the patatin gene or the 1-SST gene fromAspergillus foetidus (Rehm et al., J. Bac. 180 (1998), 1305-1319),allowing secretion of the fructosyltransferase into the medium. Cellsare advantageously used that possess a reduced or no secretory invertaseactivity at all. Fungal species with no invertase activity of their ownare, e.g., Trichoderma reesei. A protocol for the expression of aβ-fructofuranosidase (the invertase from A. niger) has been described,e.g., in Bergès et al. (Curr. Genet. 24 (1993), 53-59). A nucleic acidmolecule according to the invention which encodes a protein havingfructosyltransferase activity, or a corresponding vector, may betransfected or transformed with the host cell by conventional techniquesby the person skilled in the art. Processes for producing fused,operably linked genes and their expression in suitable host cells arewell-known to those skilled in the art and have been described, e.g., inSambrook or Ausubel, see above. Preferred hosts are E. coli, fungi,particularly yeasts, and plant cells.

The cells according to the invention are preferably characterized inthat the nucleic acid molecule introduced is heterologous with respectto the transformed cell, i.e., that it does not naturally occur in thesecells, or that it is localized in a locus of the genome different fromthat of the corresponding naturally occurring sequence.

When the nucleic acid molecules according to the invention are expressedin plants it is generally possible that the synthesized protein islocalized in any compartment of the plant cell. In order to achievelocalization in a specific compartment, the nucleic acid moleculeaccording to the invention may be linked to DNA sequences that ensurelocalization in the desired compartment; see above. Such sequences areknown (see, e.g., Braun, EMBO J. 11 (1992), 3219-3227; Wolter, Proc.Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald, Plant J. 1 (1991),95-106; Rocha-Sosa, EMBO J. 8 (1989), 23-29).

The hosts according to the invention thus comprise transgenic plantscells, plant tissues and plants that are transformed with one or severalnucleic acid molecule(s) according to the invention, as well astransgenic plant cells that originate from cells transformed in thismanner. Such cells contain one or several nucleic acid molecule(s) whichis (are) preferably linked with regulatory DNA elements allowingtranscription in plant cells, particularly with a promoter. Such cellscan be distinguished from naturally occurring plant cells in that theycontain at least one nucleic acid molecule according to the inventionwhich does not naturally occur in these cells or in that such a moleculeis localized in a locus of the genome of the cells where it is notnaturally localized, i.e., in different genomic surroundings.

In another embodiment, the present invention relates to plant cellscontaining in their cytosol the protein according to the invention. Forthis embodiment, the sequence indicated as SEQ ID No. 2 is to be usedwithout further signal sequences.

In another preferred embodiment, the present invention relates to plantcells containing the protein according to the invention in theirplastids.

In order to bring about a plastidic localization of the proteinaccording to the invention, one may modify the nucleic acid moleculesaccording to the invention and/or the vectors according to the inventionin the above-described manner.

Since the vacuole is usually capable of storing large amounts ofsucrose, which serves the protein according to the invention assubstrate, this compartment is perfectly suitable to generate plantcells which due to the activity of a protein according to the inventionproduced polyfructose in the vacuoles.

In a particularly preferred embodiment, the present invention thereforerelates to plant cells containing in the vacuole the protein accordingto the invention.

It was already explained above how the nucleic acid molecules and/orvectors according to the invention have to be constructed to mediate alocalization of the protein according to the invention in the vacuole.

The transgenic plant cells and plant tissues may be regenerated to wholeplants by methods known to those skilled in the art. The plantsobtainable by regeneration of the transgenic plant cells according tothe invention are also the subject matter of the present invention.Another subject matter of the present invention are plants that containthe transgenic plant cells described above. The plants according to theinvention may generally be plants of any plant species, preferably theyare monocotyledonous or dicotyledonous plants. Preferably, the plantcells originate from agriculturally useful plants, i.e., plants that arecultivated by man for the purpose of provision with food or fortechnical, particularly industrial purposes. Preferably, the inventionsrelates to fiber-producing (e.g., flax, hemp, cotton), oil storing(e.g., rape, sunflower soy bean), sugar-storing (e.g., sugar beet, sugarcane, sweet sorghum, banana) and protein-storing plants (e.g.,leguminbsae).

In another preferred embodiment, the invention relates to forage plants(e.g., fodder or forage grasses, alfalfa, clover, etc.), vegetables(e.g., melon, tomato, banana, chicory, leek, asparagus, carrots) orstarch-storing plants (wheat, barley, oats, rye, potato, maize, rice,pea, cassava, mungo bean).

In another embodiment, the invention relates to plant cells fromsucrose-containing plants (e.g., sugar beet, potato, rice, wheat, sugarcane, etc.). Particularly preferred are sugar beet, chicory, rice,maize, potato, sugar cane and wheat. The invention also relates to thepropagation material and harvest products of the plants according to theinvention, for example, fruits, seeds, tubers, root stocks, seedlings,cuttings, calli; cell cultures, etc.

The present invention also relates to processes for producing transgenicplants wherein

-   (a) a plant cell is genetically modified by introducing a nucleic    acid molecule according to the invention and/or a vector according    to the invention; and-   (b) a plant is regenerated from the cell; and optionally-   (c) further plants are generated from the plant according to (b).

In the context of the present invention, the term “genetically-modified”means that the plant cell is modified in its genetic information due tothe introduction of a nucleic acid molecule according to the inventionand that the presence or the expression of the nucleic acid moleculeaccording to the invention results in a phenotypic modification.Phenotypic modification preferably means a detectable modification ofone or several functions of the cells. For example, genetically modifiedplants according to the invention exhibit an activity of the proteinaccording to the invention or an increased overall fructosyl transferaseactivity.

The plants can be regenerated according to step (b) according to methodswell-known to the person skilled in the art.

The generation of further plants according to step (c) of the processesaccording to the invention may be done, for example, either vegetatively(for example using cuttings, tubers or by callus culture andregeneration of whole plants) or generatively. Generative propagationpreferably proceeds in a controlled manner, i.e., selected plants havingspecific properties are cross-bred and propagated.

The present invention relates to the plants obtainable by the processesaccording to the invention.

The present invention also relates to the propagation material of plantsaccording to the invention as well as the transgenic plants generated bythe processes according to the invention. The term “propagationmaterial” comprises those parts of the plant that are suitable forproducing successors either by the vegetative or generative route. Forvegetative propagation, e.g., fruits, seed, seedlings, protoplasts, cellcultures, etc. Preferably, the propagation material are tubers andseeds.

In another embodiment, the present invention relates to harvestableplant parts of the plants according to the invention, such as fruits,leaves, storage roots, roots, flowers, buds, sprouts or stems,preferably seeds or tubers.

In another preferred embodiment, the present invention relates tofoodstuff for animals and/or humans which contain the harvestable plantparts according to the invention, preferably sees or tubers.

Preferably, the plant parts according to the invention, afterconsumption, have an advantageous effect on the health of humans and/oranimals as compared to the corresponding parts of plants that have notbeen genetically modified in the manner described in the invention. Thesame applies to the foodstuff for animals and/or humans described in theinvention. In humans, the consumption of the foodstuff according to theinvention may, for example, lead to an improved composition of theintestinal flora, particularly to an increase in the content of bifidobacteria in the intestine, which is presumed to have a positive effecton human health (Izzo, Trends in Food Science & Technology 9 (1998),255-257). These positive effects are preferably prophylactic effects oreffects supporting the utilization of the foodstuff.

Another subject matter of the invention are processes for producing hostcells, whereby suitable host cells are transformed with a nucleic acidmolecule or vector according to the invention. Processes for thetransformation of the various host cells to be contemplated are known tothe person skilled in the art.

In another embodiment, the present invention relates to processes forproducing a fructosyltransferase, whereby a host according to theinvention is cultivated under conditions sufficient for the expressionof the nucleic acid molecule according to the invention and then thefructosyltransferase is isolated from the culture, i.e., the cellsand/or the possibly present culture medium. In the above-mentionedprocess, the transformed or transfected host cells are cultivated, forexample, in fermenters until an optimum cell density is reached.Optionally, in the case of inducible promoters, expression of theprotein encoded by the nucleic acid molecule according to the inventionis induced only at the end of the fermentation step. The proteinexpressed in this manner can then be purified from the medium, celllysates or cellular membrane fractions according to conventionaltechniques. The proteins which have been expressed, e.g., microbially,may be isolated and purified by preparative chromatographic orimmunological purification methods, for example, by using monoclonal orpolyclonal antibodies which recognize the protein encoded by the nucleicacid molecule according to the invention. In this context it should bementioned that the protein having fructosyltransferase activity andbeing encoded by the nucleic acid molecule according to the inventionmay contain additional functional amino acid sequences, for exampleprotein tags (GST, GFP, Flag, HA peptide, His-tag) that may originatefrom heterologous proteins or may have been synthetically produced.

The invention furthermore relates to proteins possessingfructosyltransferase activity, i.e., fructosyltransferases encoded bythe nucleic acid molecules according to the invention or obtainable bythe method according to the invention. The fructosyltransferasesaccording to the invention may preferably be used to producepolyfructoses of the inulin type. They may also serve to produceantibodies which may be used to detect and/or purifyfructosyltransferases.

Another subject matter of the invention are nucleic acid molecules thatspecifically hybridize to the nucleic acid molecules according to theinvention or fragments thereof. These molecules are preferablyoligonucleotides with a length of at least 10, particularly 15 andparticularly preferred at least 50 nucleotides. The oligo-nucleotidesaccording to the invention may, e.g., be used as primers for a PCRreaction, as hybridization probes or the like.

Another subject matter of the present invention are processes forproducing polyfructoses, particularly of the inulin type, whereby hostcells according to the invention, or host organisms containing them, arecultivated under conditions allowing expression of thefructosyltransferase according to the invention as well as synthesis ofpolyfructose.

By the provision of the nucleic acid molecules according to theinvention it has become possible to produce—by way of methods of genetechnology—polyfructoses, particularly of the inulin type, in organisms,such as has not been possible so far using conventional methods. It isthus possible to express the nucleic acid molecules according to theinvention in hosts such as bacteria, fungi or plant cells in order toincrease the activity of the corresponding fructosyltransferase or tointroduce it into cells that normally do not express said enzyme. Due tothe expression or additional expression of at least one nucleic acidmolecule according to the invention the host cells according to theinvention synthesize polyfructose, particularly of the inulin type.Another subject matter of the present invention are therefore thepolyfructoses, particularly of the inulin type, obtainable from the hostcells according to the invention as well as obtainable from thepropagation material and, for plants, obtainable from the plants andtheir harvest products.

Thus, the present invention particularly relates to, the production ofpolyfructoses, particularly of the inulin type, comprising:

-   (a) cultivating a host cell according to the invention, or a host    containing such a cell, under conditions allowing the production of    fructosyltransferase and reaction of sucrose, optionally added from    outside the cell, or a substrate equivalent to polyfructoses of the    inulin type; and-   (b) obtaining the polyfructose produced in that manner from the    cultivated host cells, hosts, or from the medium.

The host cells preferably are plant cells and the hosts are preferablyplants. A method for obtaining polyfructose, particularly of the inulintype, from plants is described in, e.g., Vogel (in: Inulin andInulin-containing Crops, Elsevier Science Publishers B. V. Amsterdam, A.Fuchs (Ed.) (1993), 65-75).

Another subject matter of the present invention is an in vitro processfor producing polyfructose, particularly of the inulin type, comprising:

-   (a) contacting sucrose or an equivalent substrate with a    fructosyltransferase according to the invention under conditions    allowing the conversion to polyfructose; and-   (b) obtaining the polyfructose produced in that manner.

A substrate equivalent to sucrose is, for example, a substrate that isconverted to sucrose by the host cell or one or several other enzyme(s).A substrate equivalent to sucrose may also be those di- oroligosaccharides which may be alternatively used as substrate by thefructosyltransferase according to the invention. An example of thesesaccharides is the trisaccharide raffinose. However, derivatized sucrosemay also be used. Preferably, the inulin obtained according to theabove-mentioned process is a long-chain inulin and preferably has apolymerization degree of DP>20, preferably of DP>50, particularly ofDP>100, and particularly preferred a polymerization degree of DP>200.

The present invention furthermore relates to a process for producingpolyfructose, particularly of the inulin type, comprising the step ofextracting the polyfructose from one of the above-described plants/plantcells and or from parts of such plants according to the invention.Preferably, such a process also comprises the step of harvesting thecultivated plants and/or parts of these plants prior to extracting thepolyfructose and particularly preferred the step of cultivating theplants according to the invention before harvesting them. Processes forextracting the polyfructose from plants or parts of plants are known tothose skilled in the art and have been described by, e.g., Gibson(International Sugar Journal 96 (1994), 381-387), Vogel (Stud. PlantSci. 3 (1993), Inulin and Inulin-Containing Crops, 65-75).

Also, the present invention relates to polyfructose, particularly of theinulin type, which is obtainable from the host cells according to theinvention or according to a process according to the invention describedabove. This polyfructose may preferably be used for producingsurfactants, for increasing the viscosity of aqueous systems, asdetergent, as suspension agent for sedimentation acceleration andcomplexing, or to bind water.

Also, the host cells according to the invention which synthesizepolyfructose, particularly of the inulin type, may be used as foodadditives. Such use is advantageous since fructans have positive effectson health. (Roberfroid et al., J. of Nutrition 128 (1998), 11-19;Kleesen et al., Am. J. Clin. Nutr. 65 (1997), 1397-1402).

The present invention furthermore relates to a process for producingpolyfructose, particularly of the inulin type, characterized in that afungal fructosyltransferase is used for producing the polyfructose, or ahost organism expressing a fungal fructosyltransferase. Preferably,fructosyltransferases according to the invention or host cells accordingto the invention can be used. The present invention shows for the firsttime that it is possible to use such fungal fructosyltransferases forproducing polyfructose of the inulin type.

Finally, the present invention relates to the use of fungalfructosyltransferases for producing polyfructose, particularly of theinulin type.

These and other embodiments are disclosed and obvious to the personskilled in the art and are comprised by the description and the examplesof the present invention. Further literature on one of the methods,media and uses described above which may be utilized in the presentinvention can be taken from the prior art, e.g., from public libraries,e.g., using electronic media. For this purpose, public data bases suchas “Medline” are useful which are available via internet, e.g., underthe address http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Furtherdata bases and addresses are known to the person skilled in the art andcan be found in the internet, e.g., under the addresshttp://www.lycos.com. An overview of the sources and informationregarding biotech patents or patent applications is provided in Berks,TIBTECH 12 (1994), 352-364.

FIG. 1 shows the construction of pSK-as1 for the transformation ofbacteria.

FIG. 2 shows the construction of plasmid p112-as1 for the transformationof yeast cells.

FIG. 3 shows the construction of plasmid pA7-as1 for the transformationof plant cells.

FIG. 4 shows the construction of plasmid p35-as1 for the transformationof plants.

FIG. 5 shows the construction of plasmid p35-s3-as1 for thetransformation of plants.

FIG. 6 shows an analysis by thin-layer chromatography of an E. colitransformed with pSK-as1. Lane 1 shows a control experiment with thevector without insert pBiuescript SK Lane 2 shows an experiment withplasmid pas1 in which the as1 coding region is not in frame with thelacZ gene (lane 2). In this case, translation of the as1 coding regionis not carried out as fusion to the β-glucuronidase but proceeds, withreduced efficiency, starting from the endogenous start codon. Lanes 3 to6 show experiments with various transformants of construct pSK-as1.After growing the bacteria up to an OD 600 of 0.4, the cultures wereinduced with 100 mM IPTG. After two hrs induction, the cells wereharvested and lysed in 50 mM sodium phosphate pH 6.0. Protein extractswere incubated for 12 hrs with 600 mM sucrose at 37° C. As standard forthin-layer chromatography, 1-kestose (7), sucrose (8) and fructose (9),respectively, were applied in lanes 7-9.

FIG. 7 shows an analysis by thin-layer chromatography of transformedyeast cells containing plasmid p112-as1 (lane 2) or p112-as1L (lane 3).Vector p112-as1L contains the 5′-leader of the sucrose transporter fromspinach. Lane 1 shows a control experiment with non-transformed yeastcells. Fructosyltransferase activity was detected in protein extractsfrom yeast cells. As standard, fructose (lane 4), a mixture of1-kestose, nystose and fructosyl-nystose (lane 5) and sucrose (lane 6)was applied.

FIG. 8 shows an analysis by thin-layer chromatography of plant cellscontaining plasmid pA7-as1. Lane 1 shows the transformation with thevector without insert pA7 (50 μg); lanes 2 to 5 show transformationswith vector pA7-as1 (lane 2: 10 μg; lane 3: 20 μg; lanes 4 and 5: 50μg). As standard, a mixture of 1-kestose, nystose and fructosyl-nystose(lane 6), sucrose (lane 7) and fructose (lane 8) was applied.

For every experiment 500,000 protoplasts were used. The protoplasts wereincubated for two days at 25° C. after transformation, then a proteinextract was obtained in 50 mM sodium-phosphate pH 6 which was incubatedfor 20 hours at 28° C. with 500 mM sucrose. 4 μl of a 1/10-dilution ofthe mixture were applied.

FIG. 9 shows an analysis by thin-layer chromatography of plants thatwere transformed with the construct 35-as1. Twelve plants were selectedat random. 20 mg leaf material each were extracted in 200 μl water. 4 μlof the extract were applied. As standard, fructose (lane F), sucrose(lane S) and a mixture of 1-kestose, nystose and fructosyl-nystose (laneSt) were applied.

FIG. 10 shows an analysis by thin-layer chromatography of plants whichwere transformed with the construct 35-S3as1. Twelve plants wereselected at random. 20 mg leaf material each were extracted in 200 μlwater. 4 μl of the extract were applied. As standard, fructose (lane F),sucrose (lane S) and a mixture of 1-kestose, nystose andfructosyl-nystose (lane St) were applied.

FIG. 11 shows the eluate “400 to 0 mM ammonium sulfate” of a phenylsuperose column which was loaded with a protein extract from Aspergillussydowi enriched with fructosyltransferase (lane E). In the lanescharacterized by “M” a size marker is separated. The molecular masses ofthe marker proteins are indicated on the right hand in kDaltons.

The examples illustrate the invention.

EXAMPLE 1

Purification of the af1-SST from Aspergillus sydowi

Aspergillus sydowi IAM 2544 was grown on a culture medium that contained2% malt extract, 0.5% peptone and 2% sucrose. The medium was solidifiedby adding 2% agar. Spores were plated and the culture was maintained at25° C. until the plates were completely dried. Conidia were harvestedfrom the plates and dissolved in 50 mM sodium phosphate pH 6.0. Lysis ofthe conidia was performed by three passages trough a “French PressureCell”.

For purification, the homogenate was adsorbed onto Sepharose Q. Boundprotein was eluted with a linear gradient of 0 to 1000 mM KCl.Sucrolytically active fractions were obtained between 500 and 700 mMKCl. These fractions were pooled and dialyzed against sodium phosphatepH 6.0. For enriching the protein, it was again adsorbed onto SepharoseQ (bed volume 2 ml) and eluted in a volume of 10 ml.

The eluate was adjusted to 2 M ammonium sulfate and adsorbed to phenylsuperose. Elution was carried out after washing with 2 M ammoniumsulfate, 100 mM sodium phosphate pH 7.0 with a linear gradient of 2 M to0 M ammonium sulfate. Active fractions were obtained in the elutiongradient between 400 to 0 mM ammonium sulfate. The protein mixtureobtained was analyzed by SDS-PAGE. The result is shown in FIG. 11. Asimilar enrichment of a sucrolytic activity—however, with a mycelium ofAspergillus sydowi—was described by Muramatsu and Nakakuki (BiosciBiotech Biochem 59 (1995), 208-212). Purification does not yield ahomogenous protein which would be suitable for, e.g., sequencing.

For an identification of the fructosyltransferase, a semi-nativepolyacrylamide gel was used onto which 10 μg protein of the eluate ofthe phenyl superose column in 0.1% SDS, 10% glycerol, 50 mM Tris pH 6.8were applied. After electrophoresis the gel was rebuffered three timesfor 10 minutes in 50 mM sodium phosphate pH 6.0, 1% Triton×100 and thenincubated for 30 minutes in 50 mM sodium phosphate pH 6.0, 1%Triton×100, 500 mM sucrose. Then the gel was boiled in 0.1% (w/v)2,3,5,-triphenyltetrazoliumchloride (TTC), 0.5 M NaOH. TTC thereby formsa red formazan dye together with reducing sugars. The protein bandlabeled in FIG. 11 resulted in a stain due to the sucrolytic activity ofthe protein, which could thereby be identified as fructosyltransferaseof Aspergillus sydowi. The band was isolated from a preparative gel, theprotein was eluted from the gel and used for sequencing. Since theprotein is N-terminally blocked, cleavages with endopeptidase LysC andAspN performed, the peptides were purified by HPLC and subjected to asequencing according to Edmann. The following sequences were obtained:LysC: VLPSTSQASEK (SEQ ID No. 3) AspN: DDLVTYR (SEQ ID No. 4) DPYVFQNHEV(SEQ ID No. 5)

Fore cloning the gene, a cDNA library was constructed in phage LambdaZap II (Stragene, Heidelberg). Since it was not possible to prepare RNAfrom the conidia, RNA was prepared from the mycelium according toLogemann et al. (Anal. Biochem. 163 (1987), 16-20). Poly A⁺-RNA wasobtained by the polyATract System (Promega, Madison, USA). Synthesis ofcDNA and cloning in Lambda Zap II was carried out following themanufacturer's instructions (Stratagene, Heidelberg). In accordance withthe protein sequences the following primers were designed: Primerasp19down: 5′-GAYGAYYTNGTNACNTAYMG (SEQ ID No. 6) Primer asp19up:5′-CKRTANGTNACNARRTCRTC (SEQ ID No. 7) Primer asp31-down:5′-GTNTTYCARAAYCAYGARG (SEQ ID No. 8) Primer asp31up:5′-TGRTTYTGRAANACRTANGG (SEQ ID No. 9) Primer lys1up:5′-GCYTGNSWNGTNSWNGG (SEQ ID No. 10)

In a PCR reaction with the entire cDNA library as matrix and the primercombination asp19down/asp31 up (annealing temperature 40° C.) a DNAfragment of about 350 bp was obtained. Said fragment was used afterradioactive labeling (Megaprime Kit, Boehringer Mannheim, Mannheim) forscreening the cDNA library. Clones obtained were amplified after in vivoexcision as pBluescript plasmids. The cDNA inserts were compared afterrestriction cleavage and the inserts of a clone were completelysequenced. The sequence of the cDNA insert is shown in SEQ ID No 1. Thederived protein sequence is shown in SEQ ID No 2.

EXAMPLE 2

Production of constructs containing coding regions of fungalfructosyltransferases for the transformation of various pro- andeukaryotic host cells.

For the transformation of various host cells with fungalfructosyltransferases a number of different constructs was preparedaccording to molecular-biological standard techniques (Sambrook et al.,1989, Cold Spring Harbor Laboratory Press). The constructs are shown inFIGS. 1 to 5. Specifically, the constructs were prepared as follows:

-   pSK-as1 is a derivative of pas1 which was obtained as in vivo    excision from a Lambda Zap II clone of the cDNA library of    Aspergillus sydowi. pas1 contains the cDNA as EcoRI/XhoI fragment.    pSK-as1 results from pas1 by cleavage of BamHI and SmaI, filling in    the cohesive BamHI end and religating the vector. By removal of 4    nucleotides the coding region of as1 is switched to the reading    frame of the IacZ gene (FIG. 1).-   p112-as1 Into vector p112A1NE (see Riesmeier et al., EMBO J. 11    (1992), 4705-4713), which had been cleaved with BamHI, filled in and    then cleaved with NotI, fragment as1 from pas1 (cleaved with Asp718,    filled in and cleaved with NotI) was cloned (FIG. 2).-   pA7-as1 was generated from pA7 by cloning the coding region of pas1    as SmaI/Asp718 fragment, the cohesive ends of which had been filled    in, into the filled-in Asp718 and SmaI restriction site of the    vector. The correct orientation of the fragment was confirmed by a    HindIII cleavage which resulted in an about 1900 bp fragment. pA7 is    a derivative of pUC18, which contains between EcoRI and Asp718 the    35S RNA promoter of the cauliflower mosaic virus (CaMV; 528 bp; nt    6909-7437, Franck et al., Cell 21 (1980), 285-294), as well as    between SphI and HindIII the terminator of the octopin synthase gene    from Agrobacterium tumefaciens (Gielen et al., EMBO J. 3 (1984),    835-846) (FIG. 3).-   p35-as1 was generated from pBinAR by ligating fragment as1 from pas1    (cleaved with Asp718/NotI and then filled in) into the vector which    had been cleaved with SmaI. pBinAR is a derivative of pBin19 (Bevan,    Nucl. Acids Res. 12 (1984), 8711) which contains between EcoRI and    Asp718 the 35S RNA promoter of the cauliflower mosaic virus (CaMV;    528 bp; nt 6909-7437, Franck et al., loc. cit.), as well as between    SphI and HindIII the terminator of the octopin synthase gene from    Agrobacterium tumefaciens (Gielen et al., loc. cit.) (FIG. 4).-   p35-s3-as1 was cloned in two steps. First, a BamHII Asp718 fragment    from pas1, the cohesive ends of which had been filled in with T4    polymerase, was cloned into vector pS3, which had been cleaved with    BamHI and then filled in. Thereby, pS3-as1 was obtained. Vector pS3    contains a PCR fragment of the patatin gene B33 (Rosahl et al., Mol.    Gen. Genet. 203 (1986), 214-220) which comprises nucleotides 725    to 1400. The PCR fragment is provided with an Asp718 restriction    site (GGTACC) at nt 725, with an ATGG sequence at nt 1400, which in    combination with the nt 1399 and 1400 gives an NcoI restriction    site. The PCR fragment is inserted between the Asp718 and the SmaI    restriction site. From pS3-as1 a SacI (filled in)/XbaI fragment was    prepared which contains the fusion S3-as1. This fragment was cloned    between the SmaI and the XbaI restriction site of pBinAR (FIG. 5).

The corresponding hosts were transformed by standard techniques. E coliwas transformed according to the method of Hanahan (J. Mol. Biol. 166(1983), 557-580), Saccharomyces cerevisiae was transformed according tothe method by Dohmen et al. (Yeast 7 (1991), 691-692), transient geneexpression in tobacco protoplasts was carried out according to themethod by Damm and Willmitzer (Mol. Gen. Genet. 213 (1989), 15-20),stable transformation of potato plants according to the method by Dietzeet al. (in: Potrykus, I. and G. Spangenberg (Ed.). Gene transfer toplants. xxii+361 (1995), 24-29; Springer-Verlag: Berlin, Germany; NewYork, N.Y., USA. ISBN 3-540-584064).

EXAMPLE 3

Analysis of the fructosyltransferase activity of transgenic host cellsor organisms expressing, fungal fructosyltransferases.

In Vivo Synthesis of Inulin

Transgenic host cells or organisms which express fungalfructosyltransferases were cultured in media with 2% sucrose—unless itwas plant tissue. In the case of Escherichia coli K12 as host organism,a functional cscB gene encoding the sucrose permease of E. coli wasintroduced as construct into the vector pACYC184. In the case ofSaccharomyces cerevisiae the gene of the sucrose transporter of spinachwas introduced into the vector p112AINE (Riesmeier et al., EMBO J. 11(1992), 4705-4713). The cells were cultured for at least 24 hrs in thepresence of sucrose, then harvested and broken up after washing in 50 mMsodium phosphate pH 6.0.

Plants expressing the fungal fructosyltransferases were grown in soil.After four weeks leaf and other tissue samples were taken and extractedin 1 ml water/g fresh weight in the presence of insolublepolyvinylpolypyrrolidone, cell debris was removed by centrifugation.

4 μl each of the extracts were applied on silica gel on pre-cast DCfilms (Schleicher and Schüll, Dassel, Germany) and developed twice inacetone/water (87:13). The assay for fructosyl residues was carried outwith an urea-phosphoric acid reagent (Röber et al., Planta 199 (1992),528-536).

In Vitro Synthesis of Inulin:

Cells expressing fungal fructosyltransferases were broken up in 0.50 mMsodium phosphate pH 6.0, 50 μM PMSF, 1 mM DTT, 10% (v/v) ethyleneglycol. Extracts of the cells were incubated in 50 mM sodium phosphatepH 6.0, 500 mM sucrose, 50 μM PMSF, 1 mM-DTT, 0.02% (w/v) NaN₃, 10%(v/v) ethylene glycol for 12 hrs at 25° C. The mixtures were diluted1:10 in water, then 4 μl were applied on pre-cast silica gel DC films(Schleicher and Schüll, Dassel, Germany) and developed twice inacetone/water (87:13). The assay for fructosyl residues was carried outwith an urea phosphoric acid reagent (Röber et al., loc. cit.).

The results of the individual analyses are shown in FIGS. 6 to 10. TheFigures show silica gel films after thin-layer chromatography ofincubation mixtures or cell homogenates and staining withfructose-containing sugar. By way of thin-layer chromatography inacetone/water (87:13) the carbohydrate mono-, oligo- and polymers areseparated according to size. Fructose migrates further than sucrosewhich in turn migrates further than kestose, etc. Oligomers of a DP>7and more are not separated and remain at the site of application.

In FIG. 6 one can see that E. coli clones that are transformed with apBluescript vector without insert are not capable of converting sucrose(lane 1), while those transformed with plasmid pas1 synthesize thetrisaccharide kestose. Clones that are transformed with plasmid pSK-as1are also capable of synthesizing higher oligomers (lanes 36).Simultaneously, fructose residues are transferred to water, therebyforming fructose. Said conversion is catalyzed by the SFT fromAspergillus Sydowi. FIG. 7 shows that protein extracts of yeasts thatare transformed with plasmid 112-as1 may synthesize fructan. Thefructosyltransferase activity is higher in these yeasts than in thosewhich were transformed with the construct 112-as1L. The latter—due tothe smaller fructosyltransferase activity achieved within the timeavailable in the experiment—can only synthesize the trisaccharide. Thesize of the fructans synthesized depends on the reaction time and thefructosyltransferase activity. FIG. 8 demonstrates that the size of thefructan synthesized in the extracts of transformed tobacco protoplastsdepends at the given reaction time on the amount of fructosyltransferaseactivity achieved, which in turn depends on the amount plasmid pA7-as1transformed. In lanes 3-5 one can see that oligo- and polymers with aDP>7 have been synthesized which upon chromatography do not migrate fromthe site of application. The same holds true for plant extracts fromstably transformed plants as shown in FIGS. 9 and 10.

1-18. (canceled)
 19. A fructosyltransferase encoded by a nucleic acidmolecule comprising a nucleic acid sequence encoding a protein havingfructosyltransferase activity catalyzing the reactionn(G−F)→G−F_(n)+(n−1)G, wherein the nucleic acid sequence is selectedfrom the group consisting of: (a) a nucleic acid sequence encoding aprotein comprising the amino acid sequence of SEQ ID NO. 2; (b) anucleic acid sequence comprising the coding region of SEQ ID NO. 1; (c)a nucleic acid sequence that hybridizes to a complementary strand of thenucleic acid sequence of (a) or (b) under conditions of hybridization in50% formamide, 5×SSC, 5× Denhardt's solution, 40 mM sodium phosphate pH6.8, 0.5% (w/v) BSA, 1% (w/v) SDS, 0.1 mg/ml herring sperm DNA at 42° C.and washing in 0.5×SSC/0.5% SDS at 60° C.; (d) a nucleic acid sequencewhich is degenerate due to the genetic code in comparison to thesequence of (c); and (e) a complement of any one of the nucleic acidsequence of (a)-(d).
 20. (canceled)
 21. (canceled)
 22. A process forproducing polyfructose comprising contacting sucrose or an equivalentsubstrate with the fructosyltransferase according to claim 19 underconditions that allow the conversion of said sucrose or equivalentsubstrate to polyfructose. 23-25. (canceled)
 26. A process for producingpolyfructose comprising using the fructosyltransferase according toclaim
 19. 27. The process according to claim 22, wherein thepolyfructose is of the inulin type.
 28. The process of claim 26, whereinthe polyfructose is of the inulin type.